ABSTRACT Neuropsychiatric disorders such as autism spectrum disorder (ASD), anxiety and depression cost hundreds of billions of dollars each year in medical, economic and social costs. Despite progress, treatments for these conditions remain limited. Hence safer, more specific drugs would have high clinical utility. One critical, drug-able target is the metabotropic glutamate receptor, mGlu5, antagonists of which robustly improve animal models of these disorders; however the results of initial clinical trials have been mixed. Rather than invalidating mGlu5 as a therapeutic target, such results highlight the need for a better understanding of mGlu5 function, including its subcellular localization. For example, we have shown that 70-90% of mGlu5 is located on intracellular membranes, where it couples to signaling systems distinct from those of its cell surface counterpart. Importantly, intracellular mGu5 is sufficient for establishing long term depression (LTD), a form of synaptic plasticity that is dysfunctional in ASD, anxiety and depression. The objective of the proposed research is to establish the role that intracellular mGlu5 plays by identifying differences in its function on the cell surface vs. inside the cell, in vitro, ex vivo and in vivo. Our central hypothesis is that mGlu5 interacts with different signaling pathways depending on where it is located and that this results in distinct regulation of synaptic processes and related behaviors. To test this hypothesis we have developed a genetically restricted mouse line in which mGlu5 is present only on intracellular membranes (mGlu5IM). We will use this newly derived animal line in the following aims. In Aim 1, we will determine whether mGlu5IM vs. mGlu5WT or mGlu5KO has different effects on signaling pathways in vitro (cultured neurons) by testing proposed candidate genes and by using unbiased transcriptome profiling (RNA-Seq) to identify new pathways. In Aim 2, we will use ex vivo slice preparations to determine whether mGlu5IM vs. mGlu5WT or mGlu5KO variants play unique roles and signal through distinctive pathways in synaptic processes such as LTD. In Aim 3, we will determine whether the variant mice exhibit unique effects on sensorimotor, anxiety-like and depressive-like behaviors as well as on operant models of reward. This would be the first report of location- specific functions of intracellular mGlu5 in vivo. As mGlu5 is one of a growing number of receptors that signal from inside the cell, the proposed experiments will also enhance knowledge of other intracellular receptors. Future studies targeting drugs to intracellular vs. cell- surface-localized receptors are expected to lead to the development of better drugs for mGlu5- modulated disorders such as ASD and depression,